Differential Genomic and Proteomic Signatures In Cord Blood (CB) Vs Peripheral Blood (PB) CD56+Dim NK Cells: Over Expression of CD34 In CB Vs PB CD56+Dim NK Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2781-2781
Author(s):  
Nancy S Day ◽  
Evan Shereck ◽  
Janet Ayello ◽  
Catherine McGuinn ◽  
Prakash Satwani ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Cord Blood ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Over Expression ◽  
Apoptotic Genes

Abstract Abstract 2781 Background. Umbilical cord blood (UCB) is a viable alternative source of allogeneic hematopoietic stem cells for the treatment of both malignant and non-malignant disease (Cairo et al BBMT 2008). UCB transplantation (UCBT) is known to be associated with decrease severe acute graft-versus-host disease (GvHD) compared to unrelated bone marrow (BM) and peripheral blood (PB) transplantation; however, it is associated with delayed hematopoietic and immune reconstitution (Szabolcs/Cairo et al Seminars in Hematology 2010). NK cells play important roles in both innate and adaptive immunity and are characterized as a CD56+ cell population. NK cell recovery is prompt by 2 months after hematopoietic stem cell transplantation (HSCT), while T-cell (after at least 9 mo HSCT) and B-cell (after 3 to 4 mo HSCT) reconstitutions are gradual and delayed. CD56+dim cells are primarily cytotoxic and make up 90% of PB NK populations (Shereck/Cairo PBC 2007). We previously demonstrated the ability to ex-vivo expand CB MNC into various phenotypes of CD56+dim and CD56+bright NK cells (totally 60%) and NKT cells (40%) with profound in vitro and in vivo cytotoxicity against hematological malignancies (Ayello/Cairo BBMT 2006 & Exp. Hematology 2009). Proteomic studies from our group demonstrated differential protein expression including ↑NKG2A, ↓IP3R type 3, ↓MAPKAPK5, and ↑NOTCH 2 in CB vs PB CD56+dim (Shereck/Cairo, ASH 2007; Shereck/Day/Cairo, ASBMT 2009). Objective. In these studies, we sought to determine the similarity or differences in genetic signatures in CB vs APB CD56+dim NK cells. Methods. CB MNCs were isolated on a ficoll gradient and NK CD56+16+dim cells isolated using a 2-step magnetic activated cell separation (MACS) process via a standard kit (Miltenyi Biotec). Enrichment was at least 94%. Isolated RNA from CB and PB CD56+dim cells were subjected to microarray studies (Affymetrix, U133A_2) as we have previously described (Jiang/Cairo et al J Immunol 2004). Data were analyzed by Agilent GeneSpring and Ingenuity pathway analyses. Welch test were used to perform statistical analysis and fold change of < 1.5 and values of p<0.05 were considered to be significant. Two-color ECL Plex fluorescence Western blotting (WB) was preformed to validate the proteomic data. Protein samples were separated using SDS-PAGE followed by transblotting. WB membranes were then incubated with target and control (GAPDH) primary antibodies. After rinse and wash, the membranes were further incubated with CY5 and CY3 conjugated secondary antibodies. The membranes were scanned with TYPHOON by green (532 laser and 580 filter) and red (633 laser and 670 filter) setting for CY3 and CY5, respectively, and then observed and quantified using ImageQuant. Results. CB vs PB CD56+dim cells significantly altered expressed 796 genes, in which 486 genes were over expressed, at the genomic level including: pro-apoptotic genes: CASP10 (3.1F), TNFSF11 (4.7F), CDC2 (3.0F), BCL2L1 (4.3F), NOTCH2 (1.5F); and cell development: PBX1 (7.6F), IL1RN (5.1F), CD24 (5.3F), CD34 (3.5F), CD55 (2.1F), CCL13 (2.2F). Conversely, there was significant under expression of NF1 (5.1F), MAP2K3 (1.7F), PIK3CD (2.1F), BAX (2.9F), and JUN (2.2F). Our WB results indicate that NOTCH2 (2.4F) and PBX1 (2.2F) proteins are increased in CB vs PB CD56+dim NK cells, consistent with our proteomic results. Conclusion. These results suggest that CB vs PB CD56+dim NK are more prone to undergo programmed cell death (apoptosis) secondary to over expression of numerous pro-apoptotic genes, and may be earlier in development (pro-NK) with over expression of the CD34 gene. Furthermore, decrease CB vs PB NK cytotoxicity maybe in part secondary to increase programmed cell death in particularly increase NOTCH2 at the genomic and proteomic levels. (The first two authors contribute equally.) Disclosures: No relevant conflicts of interest to declare.

scholarly journals Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 811
Author(s):  
Pranav Oberoi ◽  
Kathrina Kamenjarin ◽  
Jose Francisco Villena Ossa ◽  
Barbara Uherek ◽  
Halvard Bönig ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

Neutropenia Does Not Delay Lymphopoietic Regeneration in NODSCIDcγ−/− Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4831-4831
Author(s):  
Stefanie Bugl ◽  
Stefan Wirths ◽  
R Müller Martin ◽  
Märklin Melanie ◽  
Tina Wiesner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Early Event ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Lymphoid Progenitors ◽  
Fate Decision

Abstract Abstract 4831 Introduction: Previously it was demonstrated that lymphopoiesis is rapidly established after transplantation of wild type stem cells into lymphopenic NODSCIDcγ−/− mice. These data were interpreted as evidence for an “empty” preformed lymphopoietic niche being replenished by lymphoid progenitors. We hypothesized that antibody-induced neutropenia might influence early post transplant fate decision to myeloid rather than lymphoid differentiation resulting in delayed lymphoid reconstitution. Materials and Methods: 25,000 flow sorted CD45.2-expressing wild type Lin-/Sca1+/c-Kit+ (LSK) cells from C57BL/6 mice were transplanted into sublethally irradiated B-/T-/NK-cell deficient NODSCIDcγ−/− mice (CD45.1). Three groups of n = 7 mice received anti-Gr1 or anti-1A8 i.p. every 48 h to induce continuous antibody-mediated neutropenia vs. PBS as control. Blood was harvested at regular intervals to monitor the engraftment. After 16, 22, and 34 days, animals were sacrificed and underwent blood and bone marrow analysis. Results: Hematopoietic regeneration started with the emergence of donor-derived monocytes in all groups as well as neutrophils in the control group as early as 9 days after transplantation. On day 14, B cells were to be detected for the first time, followed by T lymphocytes approximately 20 days after transplantation. Besides the fact that neutrophils were undetectable in the antibody treated groups, the peripheral blood revealed no significant changes between the neutropenic mice and the control group at any point of time. At the bone marrow level, an increase of LSK and granulocyte-macrophage progenitors (GMPs) at the expense of megakaryocyte erythrocyte progenitor cells (MEPs) was found in neutropenic mice. Common lymphoid progenitors (CLPs), however, were not significantly different. Conclusions: The engraftment of wild type donor cells after hematopoietic stem cell transplantation into NODSCIDcγ−/− mice started with the production of monocytes and neutrophils. B-lymphocytes were detectable by day 14 after transplantation. The production of T-cells started around day 20. Continuous antibody-mediated neutropenia did not significantly delay lymphoid regeneration. Although the marrow of neutropenic mice displayed increased proliferation of granulocyte progenitors, CLPs were unchanged. We conclude that the detection of donor-derived lymphocytes in the host peripheral blood is a relatively early event after LSK transplantation. Moreover, antibody induced neutropenia is not sufficient to induce sustainable changes in early hematopoietic fate decisions on the bone marrow level. Disclosures: No relevant conflicts of interest to declare.

KIR Ligation During Ex Vivo Expansion Allows Molding Of The KIR Repertoire

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2288-2288
Author(s):  
Dean A. Lee ◽  
Vladimir V Senyukov ◽  
Jerome R Trembley
Keyword(s):  
Nk Cells ◽  
Cell Population ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Hla Ligand ◽  
Cell Subpopulations ◽  
Hla Haplotypes

Abstract NK Cell subpopulations express tremendous diversity through polymorphisms, haplotypes, differential expression, and licensing of the Killer Immunoglobulin-like Receptors (KIR). KIR diversity affects both the predisposition to cancer, and the response to therapies such as hematopoietic stem cell transplantation. Clinical trials that take advantage of the anti-cancer properties of NK cells have been limited to choosing donors on the basis of KIR genotypes and/or HLA haplotypes. Moreover, adoptive immunotherapy approaches have been limited by low NK cell doses. The latter hurdle has been recently mitigated by methods for expanding clinical grade NK cells ex vivo. These approaches for growing large numbers of cells now enable investigation into selecting more potent NK cell subsets for increased therapeutic efficacy. We hypothesized that the desired KIR repertoire could be molded through inhibition of undesirable KIR populations by crosslinking with relevant anti-KIR antibodies during expansion with our previously described method, which produces a mean 30,000-fold expansion of NK cells in 3 weeks. First, we determined that maximum inhibition was obtained when anti-KIR antibodies were applied to previously activated NK cells, crosslinked with secondary antibody, and then restimulated for proliferation. Robust reduction of targeted KIR-positive populations could be achieved for each inhibitory KIR (Fig. 1). When pre-activated with anti-KIR2DL1 for one stimulation cycle, NK cells expressing this KIR were decreased by a median of 70.4% ± 19.3%. Similarly, KIR2DL2/3+ NK cells could be reduced by 56% ± 17.5%, and KIR3DL1+ NK cells could be reduced by 53.5% ± 16.3%. When anti-KIR antibodies were combined, similar suppression of multiple-KIR subpopulations was observed. Other NK cell receptors were not significantly affected during targeted KIR inhibition. We then assessed the resulting NK cell populations for degranulation responses to targets with selected HLA as KIR ligands. Inhibition of KIR-expressing subpopulations during expansion resulted in NK cell populations with enhanced degranulation against tumor cells expressing the HLA ligand of the targeted KIR. Importantly, the cytotoxicity of the bulk NK cell population against HLA-negative targets remained. These results indicate that KIR crosslinking during NK cell propagation enables significant reduction in the targeted KIR subpopulations, resulting in an NK cell population with a selective decrease in KIR inhibition. By utilizing antibody-controlled expansion for molding of the KIR repertoire according to patient HLA type, a personalized NK cell product may be produced with enhanced potency, improving NK cell immunotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Decreased Tim-3 Expression Level of Peripheral Blood Natural Killer Cells in Severe Aplastic Anemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4767-4767
Author(s):  
Rong Fu ◽  
Xin Yuan ◽  
Chunyan Liu ◽  
Hui Liu ◽  
Yihao Wang ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Early Stage ◽  
Bone Marrow Failure ◽  
Severe Aplastic Anemia ◽  
Killer Cells ◽  
Before And After

Abstract Severe aplastic anemia (SAA) is a life threatening disease characterized by severe pancytopenia and bone marrow failure. T-cell immunoglobulin- and mucin domain-containing (Tim)-3 has been initially used to identify dysfunctional T cells, but recent studies have demonstrated that Tim-3 is widely detected on nature killer cells (NK cell) and may serves as a marker for activation and maturation of NK cells. In our study, Tim-3, expressed on peripheral blood of NK cells in SAA patients, was quantitatively analyzed by Flow cytometry before and after immunosuppressive therapy (IST). Results showed that the expression of Tim-3 in patients before IST [(61.11±15.46)%] was significantly lower than that in patients after IST[(74.30±12.63)%, P<0.05] and normal controls[(70.39±12.73)%, p<0.05]. However, no difference was observed between patients before and after IST. Therefore, we concluded that low expression of Tim-3 in NK cells may play a crucial role in early stage of SAA. Disclosures No relevant conflicts of interest to declare.

CD16+CD56− NK Cells in the Peripheral Blood of Cord Blood Transplant Recipients: A Unique Subset of Immature NK Cells Possibly Associated with Graft-Versus-Leukemia Effect.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1074-1074 ◽  
Author(s):  
Xuzhang Lu ◽  
Yokio Kondo ◽  
Hiroyuki Takamatsu ◽  
Hiroshita Yamazaki ◽  
Zhirong Qi ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cord Blood ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Flow Cytometric Analysis ◽  
Cell Subset ◽  
Leukemic Cells

Abstract NK cells play a major role in the activity of graft-versus-host (GVL) effect after an HLA-mismatched stem cell transplantation. In unrelated cord blood transplantation (CBT) where there is often an HLA mismatch between the donor and recipient, NK cells may also play a vital role, though their roles have not been extensively studied. Cord blood (CB) is known to have a unique subset of NK cells characterized by a CD16+CD56− phenotype. CD16+CD56− NK cells in CB are thought to be progenitors of CD16+CD56+ NK cells because CD16+CD56− NK cells acquires CD56 expression after in vitro culture in the presence of IL-2. However, the function of this immature NK cell subset after CBT remains unknown. A marked increase in the number of CD16+CD56- NK cells in the peripheral blood of an HLA-mismatched CBT recipient with acute myeloid leukemia (AML) was recently observed. A 56-year old male, who received a reduced intensity CBT following a full relapse after allogeneic stem cell transplantation from an HLA-matched sibling donor, showed an increase in the copy number of WT-1 mRNA in the peripheral blood around day 80 after the CBT, but the WT-1 copy number decreased from 1500/microliter RNA to 230/microliter RNA in association with the increase in the number of CD16+CD56- NK cells, and his molecular remission lasted more than 1.5 years thereafter. This case prompted an investigation of CD16+CD56− NK cells in the peripheral blood after allogeneic stem cell transplantation. A similar increase in the proportion of CD16+CD56− NK cells (20% or more) in the peripheral blood CD16+ NK cells was observed in 64% (7/11) of CBT recipients, all of whom maintained remission, but in none of the 11 bone marrow and 8 peripheral blood stem cell transplant recipients examined (Figure 1). CD16+CD56− NK cells in CBT recipients expressed receptors specific to NK cells such as NKp30 and NKp46 same level as CD16+CD56− NK cells of fresh CB cells. CD16+CD56− NK cells isolated from CBT recipients became CD56+ when they were cultured in the presence of IL-2 with or without K562-mb15-4-1BBL. When cultured NK cells derived from the CD16+CD56− NK cells were separated into CD158b+ and CD158b− cells, CD158b+ cells failed to kill 721–221 cells transfected with HLA-C*0301 while they killed untransfected or HLA-C*0401-transfected 221 cells. Despite the presence of the corresponding KIR ligand (C*0304), cultured CD16+CD56− NK cells showed cytotoxicity against the patient’s leukemic cells. These findings suggest that an increase in the proportion of CD16+CD56− NK cells is unique to recipients of CBT and that this immature NK-cell subset in CBT recipients may undergo differentiation into mature NK cells in vivo capable of killing residual leukemic cells, thereby contributing to the GVL effect regardless of the presence of the KIR ligand. Figure 1 Flow cytometric analysis of CD3-CD16+CD56-cells in peripheral blood of SCT recipients and healthy individual.Examples of three-flourescence cytofluorometric analysis of fresh isolated PBMC stained with CD3,CD56 and CD16 in different SCT patients and health individuals. The characterization of the unusual CD56-CD16+ cell subset expend only in the CBT individual(a). Presenting cellware gated on CD3-cells Figure 1. Flow cytometric analysis of CD3-CD16+CD56-cells in peripheral blood of SCT recipients and healthy individual.Examples of three-flourescence cytofluorometric analysis of fresh isolated PBMC stained with CD3,CD56 and CD16 in different SCT patients and health individuals. The characterization of the unusual CD56-CD16+ cell subset expend only in the CBT individual(a). Presenting cellware gated on CD3-cells

41BBL-Based Activation and Expansion of Autologous Natural Killer Cells Results in Enhanced Activity Against Leukemia Including ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2293-2293
Author(s):  
Ekta Kapadia ◽  
Elad Jacoby ◽  
Mark Kohler ◽  
Waleed Haso ◽  
Christopher Daniel Chien ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Enhanced Activity

Abstract Childhood leukemia is the most common pediatric malignancy. There are now excellent cure rates for these patients, however outcomes remain poor for those with refractory disease and for those who relapse after standard salvage therapies, with a disease recurrence of approximately 50%. Therefore, development of novel cellular therapies is essential to treat these refractory patients. Natural Killer (NK) cells generated from an allograft contribute to improved disease free survival after Hematopoietic Stem Cell Transplantation for leukemia when there is a KIR mismatch. This effect appears to be particularly potent in the setting of Acute Myelogenous Leukemia (AML) with less benefit demonstrated in Acute Lymphoblastic Leukemia (ALL). Preclinical studies have also suggested that activation and expansion of resting NK cells can enhance NK cell cytotoxicity and eliminate the need for KIR mismatch due to up-regulation of activating receptors. We are currently testing this approach in the clinic following a fully matched allogeneic transplant platform for leukemia. Our aim is to explore whether 41BB ligand (41BBL) and recombinant IL-15 (rIL-15) mediated ex vivo expansion of autologous NK cells results in enhanced activity against AML and ALL. The activation/expansion process may allow for the use of autologous NK cell infusions, thus eliminating the need for allogeneic NK cell donors. To test this hypothesis, we ex vivo expanded and activated NK cells derived from C57BL/6J (B6) mice using artificial Antigen Presenting Cells (aAPCs) containing 41BBL and rIL-15 for 7-14 days. NK cells were co-cultured with murine AML cells (C1498) and murine ALL cells (E2A-PBX) – both on B6 background. Controls included YAC cells (murine T-cell lymphoma cell line sensitive to NK cell killing) as well as Phorbol Myristate Acetate (PMA)/ionomycin. All cells were co-cultured for 5 hours prior to functional assessment of NK cells via CD107a degranulation. NK cells cultured with 41BBL aAPCs and rIL-15 had a 30-fold expansion in numbers (Figure 1) and an increase in purity to approximately 95-98% (NK1.1+, CD3–) by Day 7. In the absence of cytokine or aAPCs, cultured NK cells underwent rapid apoptosis. Functionally, although resting NK cells (harvested prior to assessment) expressed CD107a when cultured with YAC cells and PMA, only minimal degranulation was observed in the presence of autologous AML cells or ALL cells. In contrast, activated and expanded autologous NK cells displayed enhanced activity against ALL, AML, as well as YAC cells, while only minimal levels of CD107a were seen in the absence of targets (Figure 2). In vivo experiments with a single injection of activated and expanded NK cells did not result in prolonged survival of mice bearing either AML or ALL. Assessment of adoptively transferred NK cells demonstrated very transient persistence (<2 days) with no in vivo expansion, suggesting that repeated injections may be necessary for leukemia eradication. Future murine experiments will investigate the effect repeated injections of activated/expanded NK cells and/or the administration of rIL-15 will have on survival and leukemia eradication. In addition, the ability to activate and expand NK cells in culture provides an opportunity for lentiviral-based transduction with chimeric antigen receptor (CAR) vectors. We are currently testing this with a murine CD19 CAR. These experiments suggest that autologous activated and expanded NK cells may serve as a viable cellular therapy for pediatric patients with refractory/relapsed leukemia. As demonstrated in these in vitro experiments, autologous activated/expanded NK cells still show increased targeting of mouse AML and ALL cell lines despite the lack of KIR mismatch. Thus, they may serve as a potential platform for leukemia therapy, including ALL, which appear to be poor targets for resting NK cells. In addition, these cells demonstrate transient persistence in vivo, a potential advantage in the context of redirected cytotoxicity using CAR constructs that target antigens with broader expression in the hematopoietic compartment. Figure 1: <![if !vml]><![endif]> Figure 1:. <![if !vml]><![endif]> Figure 2: Figure 2:. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD56brightCD16- NATURAL KILLER CELLS ARE IN HIGHER COUNTS IN THE UMBILICAL CORD BLOOD THAN IN THE PERIPHERAL BLOOD

2021 ◽  
Author(s):  
Vinicius Campos de Molla ◽  
Miriam Cristina Rodrigues Barbosa ◽  
Alfredo Mendrone Junior ◽  
Matheus Vescovi Goncalves ◽  
Eliza Kimura ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Stem Cell ◽  
Natural Killer Cells ◽  
Cord Blood ◽  
Nk Cells ◽  
Umbilical Cord Blood ◽  
Peripheral Blood ◽  
Lower Incidence ◽  
Hematopoietic Stem ◽  
Classical Monocytes

Umbilical cord blood (UCB) is an alternative source for hematopoietic stem cells allogeneic hematopoietic stem cell transplantation in the absence of compatible donor. UCB transplantation has a lower incidence of chronic graft versus host disease (GvHD) but is associated with slower engraftment and slower immune reconstitution as compared to other sources. Dendritic cells (DC) and Natural Killer cells (NK) play a central role in the development of GVHD, the graft versus leukemia (GvL) effect, and in the control of infectious complications. We quantified by multiparametric flow cytometry monocytes, lymphocytes, NK cells, and DC, including their subsets, in UCB samples from 54 healthy newborns and peripheral blood (PB) from 25 healthy adult volunteers. In the UCB samples, there were higher counts of CD56brightCD16- NK cells (median 0.024x109/L), as compared to the PB samples (0.012x109/L, P<0.0001), CD56dimCD16bright NK cells (median 0.446x109/L vs. 0.259x109/L for PB samples, P= 0.001), and plasmacytoid dendritic cells (pDC, median 0.008x109/L for UCB samples vs. 0.006x109/L for PB samples, P= 0.03). Moreover, non-classical monocytes counts were lower in UCB than in PB (median 0.024x109/L vs. 0.051 x109/L, respectively, P< 0.0001). In conclusion, there were higher counts of NK cells and pDC, and lower counts of non-classical monocytes in UCB than in PB from healthy individuals. These findings might explain the lower incidence and severity of chronic GVHD although maintaining the GVL effect in UCB transplants recipients as compared to other stem cell sources.

scholarly journals The Mechanism of Activated TGF-β1 Inhibiting GVL Effects of Bone Marrow NK Cells Leading to Early Relapse after Transplantation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 48-49
Author(s):  
Dongyao Wang ◽  
Xiaoyu Zhu ◽  
Zimin Sun
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Transforming Growth Factor ◽  
Immune Escape ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Transforming Growth Factor Β1 ◽  
Hematopoietic Stem ◽  
High Level ◽  
Tgf Β1

Allogeneic hematopoietic stem-cell transplantation (allo-HSCT) is one of the best ways to cure acute myeloid leukemia (AML). However, patients with AML undergoing allo-HSCT are at risk of relapse, the mechanism remains poorly understood. Here, we demonstrated the significant decrease of nature killer (NK) cells, and the declined proportion of multi-functional effector NK cells in bone marrow from patients who had a relapse in 3 months after allo-HSCT. Furthermore, we verified the levels of activated Transforming growth factor-β1 (TGF-β1), not the total TGF-β1, increased in bone marrow of these patients. This high level activated TGF-β1 was correlated with reduced cytotoxicity of NK cell, and contributed to immune escape of tumor cells. Moreover, the expression of glycoprotein A repetitions predominant (GARP), which is critical to TGF-β1 activation, high expressed in CD4+ T cells of patients who had a relapse. These data reveal a mechanism of immune escape and proposes approaches for therapeutic administration of NK cells in order to reverse suppression of activated TGF-β1 during early allo-HSCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD38-Deficient, CD16-Engineered NK Cells Exhibit Enhanced Antibody-Dependent Cellular Cytotoxicity without NK Cell Fratricide to Augment Anti-Myeloma Immunity in Combination with Daratumumab

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3224-3224
Author(s):  
Karrune Woan ◽  
Ryan Bjordahl ◽  
Frank Cichocki ◽  
Svetlana Gaidarova ◽  
Cameron Pride ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Nk Cell ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Myeloma Cells ◽  
Cell Immunotherapy

Abstract Daratumumab targets the cell surface protein CD38 and is the only FDA approved monoclonal antibody that has demonstrated single agent efficacy in relapsed refractory myeloma. CD38 is broadly expressed in the immune system, and its high expression on multiple myeloma cells allows for effective targeting by daratumumab. Daratumumab induces myeloma cell death through multiple mechanisms, including complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and perhaps most importantly, antibody-dependent cellular cytotoxicity (ADCC). ADCC is mediated by binding of the antibody Fc region to the CD16 Fc receptor expressed on natural killer (NK) cells. Engagement of CD16 induces NK cell activation and target cell cytolysis. However, because CD38 is also expressed on the surface of NK cells, daratumumab treatment can induce NK cell fratricide, which likely impairs the effectiveness of ADCC-mediated targeting and elimination of myeloma. In addition, NK cell function is often suppressed or absent in patients with myeloma, as a result of the tumor itself or from its therapy, further reducing the effectiveness of daratumumab. Collectively, preclinical and clinical observations suggest a potential therapeutic benefit of maintaining NK cell numbers and function in patients to support daratumumab-mediated ADCC and augment the treatment of multiple myeloma. We have developed an off-the-shelf NK cell immunotherapy derived from genetically engineered, induced pluripotent stem cells (iPSC) for enhanced ADCC in combination with daratumumab. iPSCs were engineered to express a high-affinity, non-cleavable CD16 construct (hnCD16) in combination with complete bi-allelic disruption of the CD38 gene (hnCD16 CD38-/-), and the engineered iPSCs were subsequently differentiated into NK (iNK) cells. We hypothesized that CD38-deficient iNK cells would exhibit improved survival by avoiding daratumumab-induced NK cell fratricide, while expression of the hnCD16 transgene would enhance ADCC against myeloma cells in combination with daratumumab. Genetic modification was confirmed in hnCD16 CD38-/- iNK cells by flow cytometry, demonstrating abrogation of CD38 expression (Fig. 1A) and constitutive high expression of CD16 (Fig. 1B). Additionally, hnCD16 iNK cells and hnCD16 CD38-/- iNK cells expressed similar levels of SLAMF7/CD319 (the target of elotuzumab) and NKG2A (Fig. 1C and D). No significant difference in iNK cell differentiation, expansion, maturation, activation, or ability to mediate natural cytotoxicity was observed. In contrast to previous reports, we observed no effect of CD38-deficiency on CD16-mediated calcium flux between hnCD16 iNK cells and hnCD16 CD38-/- iNK cells (Figure 1E). In vitro culture of NK cells in the presence of daratumumab led to NK cell fratricide for both peripheral blood-derived NK cells and hnCD16 iNK cells (Fig. 1F). Daratumumab-induced NK cell fratricide was dependent upon expression of both CD16 and CD38, as unmodified iNK with low CD16 levels (~20% of cells) showed reduced cell death in the presence of daratumumab, which was entirely absent in hnCD16 CD38-/- iNK cells (Fig. 1F). This data was confirmed by extended culture of NK cells with RPMI-8226 tumor spheroids in the presence or absence of daratumumab. The number of hnCD16 iNK cells and peripheral blood NK cells were significantly reduced compared to hnCD16 CD38-/- iNK cells (p>0.005, Fig. 1 G). Importantly, hnCD16 CD38-/- iNK cells were better able to mediate ADCC towards MM1.S multiple myeloma cells compared to hnCD16 iNK cells (Fig. 1H). Taken together, these data support our hypothesis that targeted knock out of CD38 on NK cells alleviates daratumumab-induced NK cell fratricide that occurs through the crosslinking of CD16 and CD38 on neighboring NK cells, leading to augmented anti-myeloma immunity. These data provide a translatable, proof of concept study demonstrating precision genetic engineering of iPSC to generate off-the-shelf NK cell immunotherapy to enhance daratumumab mediated ADCC in multiple myeloma. We propose a strategy of off-the-shelf hnCD16 CD38-/- iNK infusion in combination with daratumumab to overcome NK cell depletion effects of CD38 targeted agents and to improve myeloma patient outcomes. Figure 1. Figure 1. Disclosures Bjordahl: Fate Therapeutics Inc.: Employment. Cichocki:Fate Therapeutics Inc.: Consultancy, Research Funding. Gaidarova:Fate Therapeutics Inc: Employment. Pride:Fate Therapeutics Inc.: Employment. Kaufman:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

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